Protein kinase C (PKC) consists of a heterogeneous family of isozymes derived from nine genes divided into three, classes: conventional, novel and atypical. The atypical class of isoforms (aPKC), e.g., PKCζ (PKCζI/II) and PKCι/λ, is distinguished from the conventional and novel classes by their insensitivity to calcium and diacylglycerol (DAG), the classical activators of PKC (Newton, 2003, Biochem J, 361-71). Together with the proteins encoded by par3 and par6 genes, aPKC has been shown to play a critical role in cell polarity (Ohno, 2001, Curr Opin Cell Biol, 13(5): 641-48). Recently, the par3 and par6 genes were also shown to play a role in the formation of neuronal axons (Shi et al., 2003, Cell, 112(1): 63-75). Loss of function of any of par3, par6 and aPKC proteins can result in disruption of cell polarity.
In addition, a role for aPKC in synaptic plasticity, learning and memory has been established (Sacktor et al., 1993, Proc Natl Acad Sci USA 90:8342-46; Osten et al, 1996, Neurosci Letter 221:37-40; Drier et al, 2002, Nat Neurosci 5:316-24; Ling et al, 2002, Neurosci 5:295-96). In long-term potentiation (LTP), a widely studied model for memory, aPKC plays an important role in the two distinct temporal phases. While full-length aPKC forms are activated during the LTP induction phase, a truncated form of aPKC, termed PKMζ (i.e., PKCζII), is activated during the maintenance phase of LTP. PKMζ is identical to PKCζI, except that it lacks an autoinhibitory regulatory domain (Hernandez, et al., 2003, J Biol Chem 278, 40305-16 and Hirai et al., 2003, Neurosci Lett 348, 151-54). Although all of the PKC isoforms can theoretically have PKM forms (i.e., truncated or independent catalytic domains of PKC), the only PKM form that is consistently observed in the normal brain is PKMζ (Sacktor et al., 1993). Recent work has shown that PKMζ is both necessary and sufficient for long-term potentiation (LTP) maintenance (Ling et al). Furthermore, expression of PKMζ prolongs memory in an odor avoidance task in Drosophila melanogaster (Drier et al). Thus, the role of PKMζ in memory appears conserved in widely divergent species. Consistent with this notion, the human form of the PKMζ mRNA has been identified (Hernandez, et al).
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive impairment in memory and cognitive functions. Imbalances in neuronal signal transduction pathways have been implicated in AD's pathogenesis (Shimohama et al., 1990, J Neural Transm Suppl 30:69-78). Several studies have demonstrated abnormalities of protein kinase C (PKC) function in brain tissue of AD patients (Cole et al., 1988, Brain Res 452:165-174; Clark et al., 1991, Lab Invest 64, 3544; Horsburgh et al., 1991, J Neurochem 56:1121-1129; Masliah et al., 1990, J Neurosci 10:2113-24; Masliah et al., 1991, J Neurosci 11:2759-2767; Saitoh et al., 1990, Adv Exp Med Biol 265:301-10; Saitoh et al., 1993, Acad Sci 695:34-41; Shimohama et al., 1993, Neurology 43:1407-1413; Lanius et al., 1997, Brain Res 764:75-80; Wang et al., 1994, Neurobiol Aging 15:293-298).
Microscopically, the two major features of AD are the presence of β-amyloid (A β) containing senile plaques (SP) and tau containing neurofibrillary tangles (NFT). Amyloid angiopathy, defined as amyloid deposition in blood vessels, also occurs to a varying extent. Other changes include granulovacuolar degeneration (GVD) and Hirano body (HB) formation.
The NFT are abnormal structures, composed mainly of paired helical filaments (PHFs) consisting of a hyperphosphorylated form of the microtubule-associated protein tau (Buee et al., 2000, Brain Res Brain Res Rev 33, 95-130). The distribution of tau is largely restricted to axons, where it functions mainly to stabilize microtubules (MT) and promote MT polymerization. In NFT-containing neurons, however, tau-associated PHFs can be found throughout the cytoplasm and dendrites. Other components of the NFT include microtubule-associated protein 2 (MAP2) and ubiquitin. A “ghost tangle” is a type of NFT, where the surrounding neuron has completely degenerated. Ghost tangles can contain deposits of Aβ that accumulate after cell death. PHFs also exist in dystrophic neurites surrounding the senile plaques as well as in other neurites, where they are termed neuropil threads.
In addition to AD, at least 20 different diseases have tau-based neurofibrillary pathology as a feature and are collectively known as tauopathies (Lee et al., 2001, Annu Rev Neurosci 24, 1121-59). This group includes AD, Pick's disease (PiD), frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Considerable heterogeneity exists between the tangles found in the various tauopathies. For example, NFT in AD are composed principally of PHFs with a diameter of 8-20 nm; some straight filaments are also seen in AD. In contrast, the tangles of FTDP-17 are twisted and straight, but not paired and helical. Nevertheless, the tau protein in all these disorders is hyperphosphorylated.
It has been argued that tau-based tangle formation is not the primary pathological event in AD since tangles appear in a number of other disorders. This apparent lack of specificity suggests that the NFT is a final endpoint of a number of pathophysiologic processes rather than an initiating event. Contrary to this view, it has been discovered that a mutation in the tau gene itself in multiple FTDP-17 family lineages can lead to tau dysfunction and degeneration in the absence of Aβ accumulation (Lee et al., 2001).
NFT develop through stages (Braak et al., 1994, Acta Neuropathol 87, 554-67). The earliest form of NFT is the pre-NFT, where PHFs begin to form, but the full tangle has not yet developed. The next is the intracellular stage, where the cytoplasm becomes filled with hyperphosphorylated tau. The final stage is the “extracellular” NFT, where cell death has occurred, the membrane and organelles cleared away, but the cytoskeletal remnants remain. The final stage is also referred to as “ghost” tangles. Using phosphospecific tau antibodies, it was discovered that the pattern of tau phosphorylation varies between these three different stages (Augustinack et al., 2002, Acta Neuropathol (Berl) 103, 26-35). Theoretically, if one knows which residues on tau are phosphorylated first, then one can have a better chance at identifying the kinase responsible for the tau phosphorylation. It was demonstrated that T153, S262 and T231 were among the first residues in tau to become phosphorylated stages (Augustinack et al., 2002).
Despite extensive research, the particular kinase that phosphorylates tau in AD has not been clearly identified (Buee et al., 2000). The longest tau protein isoform contains 79 possible serines and threonines, with phsophorylation of least 30 of them occurring in AD. Numerous kinases have been shown to phosphorylate tau in vitro, but leading candidate kinases have been shown to phosphorylate tau in vivo. These are glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (cdk5). Both are members of a group of proline-directed kinases that prefer serine/threonine residues directly followed by a proline. Abnormal tau from an AD brain contains a mixture of hyperphosphorylated serine and threonine residues with and without a trailing proline. Indeed, researchers have postulated multiple kinases phophorylating tau at different sites, which can be proline directed and non-proline directed protein kinases.
The conventional PKCα isoform can phosphorylate the cytoskeletal protein tau. It is not the full-length form of PKCα, but the truncated PKMα form of the enzyme which is selectively capable of phosphorylating tau (Cressman et al., 1995, M. FEBS Letter 367, 223-27). The only PKM isoform that is consistently overserved in the brain is PKMζ (Naik et al., 2000, J Comp Neurol., 426(2):243-58).
The present invention, for the first time, shows that PKMζ directly phosphorylates tau. For this reason, increases in aPKC will cause NFT formation. Furthermore, aPKC phosphorylates GSK3β, a kinase well known to phosphorylate tau, leading to inactivation of GSK3β (Lavoie et al., 1999, J Biol Chem 274, 28279-85). For this reason, decreases in aPKC activity constitute a removal of the negative regulation of GSK3β activity and indirectly causes NFT formation. The present invention provides that apkc and gsk3b, both known regulators of cell polarity, are present in a complex. Alterations in the signaling or activity of these two kinases leads to mislocalization of gene products within the intracellular and extracellular compartments as well as abnormal posttranslational modifications of gene products and subsequent pathology including neurological dysfunction and cancer.
While the etiology of cancer is heterogenous, and largely depends on the tissue type, there is a consensus among those skilled in the art that human cancer cells show a markedly increased genetic mutation rate (genetic instability). The mutations can manifest themselves as chromosomal abnormalities (e.g. deletions, insertions, amplifications, mutations and rearrangements) and point mutations. The effects of such genomic alterations are varied; but some of these alterations are direct contributors to cancer development. Cancer develops through stages. The early stages tend to be more benign, with a better prognosis. In contrast, the later stages are more malignant, and tend to metastasize to a larger extent. Late stage cancers tend to have accumulated more chromosomal alterations. The present invention utilizes alterations in the aPKC genes and their RNA or protein products to diagnose, stage, treat, and develop treatments for cancer.
One example of a chromosomal alteration associated with cancer development is deletion of the short arm of chromosome 1 (1p). Chromosome 1p deletions have been observed in a wide variety of cancers. It has been estimated that over one half of solid tumors are associated with chromosome 1p deletions. The current method for determining the status of chromosome 1p in tumors involves fluorescence in situ hybribization (FISH). While highly sensitive, FISH has numerous drawbacks including the fact that it is not routinely used in pathology laboratories, is costly and time consuming. The present invention provides a new way to determine the status of chromosome 1p in tumors using probes to the PKCζ gene, such as antisera. Antisera based methods are routinely used in pathology laboratories, are robust and inexpensive. For this reason, PKCζ gene status is by far superior to FISH in determining cancer status.
The PKCζ gene lies on the short arm of the first chromosome (1p36.33). The gene lies a relatively short distance, about 2 million base pairs, away from the telomere. The proximity of the PKCζ gene to the telomere should theoretically make it susceptible to chromosomal deletion and rearrangement. It is demonstrated by the present invention that the presence or absence of aPKC in cancers is useful for their diagnosis and staging.
The effect of deletion of chromosome 1p varies depending on the tumor type. For example, deletion of 1p in oligodendroglioma is a favorable prognostic indicator. In contrast, chromosome 1p deletion in neuroblastoma is considered unfavorable. As demonstrated by the present invention, one skilled in the art can now determine the status of chromosome 1p using specific probes to PKCζ. Examples of such probes include antisera, complimentary DNA or RNA sequences to aPKC, or PCR primers. The presence or absence of PKCζ is effective in determining the variety, stage or grade of the tumor.